1. Field of the Invention
The present invention relates to a cell-containing sheet that is useful as a medical implant, a method for producing the same, and a treatment method using the same.
2. Background Art
In recent years, a technology whereby artificial alternatives or organized culture cells are used for implantation has been under development and gaining attention. Representative examples of such alternatives or cells to be implanted include artificial skin, artificial blood vessels, and tissue composed of culture cells. In the case of artificial skin made of synthetic polymers, for example, rejection or the like might occur, therefore such skin is not preferable for implantation. Meanwhile, tissue composed of cultured cells is made by culturing and organizing cells of a patient. Thus, there is no concern about rejection when using such tissue in the patient himself/herself. Therefore, the tissue is preferable for implantation. Such tissue composed of cultured cells is produced by collecting cells from a patient and culturing the cells for the patient.
Many types of animal cells need to adhere to an object so as to proliferate, which is described as anchorage dependence. Thus, in vitro animal cells cannot live for a long period of time in a floating state. Therefore, in cell culture for producing the aforementioned tissue composed of cells, carriers, for example, a polymeric material such as modified polystyrene that has cell adhesiveness improved via surface treatment or culture dishes produced by uniformly applying a cell adhesion protein such as collagen or fibronectin to glass or a polymeric material have been used. Cells that have two-dimensionally adhered to such carriers are excellent in terms of capacity to be cultured; however, such cells are unlikely to be organized, in general. Thus, such cells lack their inherent functions. For instance, a study reported that the capacity of unorganized hepatocytes (cultured hepatocytes) to produce albumin is several times less than that of organized hepatocytes (liver spheroids).
Meanwhile, a technology has been reported whereby cultured cells are allowed to adhere to and are arranged only on minute areas on a substrate, resulting in enhanced cellular organization. In a method for arranging cultured cells, a substrate having surface patterns with different levels of ease of adhesion to cells is used, cells are cultured on the surface patterns, and the cultured cells are allowed to adhere exclusively to the surface patterns that have been processed in a manner such that cells can adhere thereto.
For instance, in JP Patent Publication (Kokai) No. 3-7576 A (1991), arrangement of cultured cells was attempted on a surface on which photosensitive hydrophilic polymers lacking or having cell adhesiveness were subjected to patterning via a photolithography method. Further, JP Patent Publication (Kokai) No. 5-176753 A (1993) discloses a substrate for cell culture on which patterns have been made using a substance such as collagen that influences cell adhesion ratio or cell form and a method for producing such substrate via photolithography. When cells are cultured on such substrate, many cells are allowed to adhere to the surface patterns formed with collagen and the like, resulting in implementation of cell patterning thereof.
When patterning is carried out by, for example, the aforementioned photolithography using a photosensitive material, extremely fine patterns can be obtained; however, cell adhesive materials are required to have photosensitivity. In such case, it is often difficult to carry out chemical modification so as to impart photosensitivity to biopolymers and the like. Thus the range of choices for cell adhesive materials is extremely narrowed, which has been problematic. In the case of photolithography using photoresists, a developing solution or the like must be used, and thus this has sometimes caused adverse effects upon cell culture. In addition, in general, biomaterials and other materials having high cell culture capacities are unlikely to be degraded via plasma, resulting in low industrial productivity upon patterning using a plasma etching method. Thus, such patterning is impractical.
Moreover, cells cultured via patterning as described above are subjected to treatment using proteinase such as trypsin or chemical agents so as to be collected. Thus, the treatment steps become complicated, resulting in a high probability of contamination. In addition, functions inherent to cells might be impaired as a result of cell degeneration or cell damage.
JP Patent Publication (Kokai) No. 2003-38170 discloses a method for producing a cell sheet, comprising the steps of: producing a cell culture support in which patterns have been formed on a substrate using temperature-responsive polymers; culturing cells on the cell culture support; allowing the cells to come into close contact with the polymer membrane; changing the temperature; and removing cells together with the polymer membrane from the support without causing damage to the cells. However, in accordance with the method disclosed in the above reference, adherence between the polymer membrane and cells is weak when cells are removed from the support together with the polymer membrane, so that it is difficult to form a cell sheet having fine patterns.
WO2005/038011 teaches a method for forming fine cell patterns on an implant site, comprising the steps of: allowing cells to adhere to the surface of a substrate for cell arrangement on which a cell adhesiveness-variation pattern containing cell adhesiveness promoted regions and cell adhesiveness inhibited regions has been developed; transferring the adhering cells in the patterned form to the implant site; and culturing the cells. However, in accordance with this method, after overlapping cells on the substrate for cell arrangement on the implant site, it is necessary to retain the substrate for cell arrangement while carrying out cell culture until cells become fixed on the implant site. Thus, it is difficult to apply such method to immediate implantation.
Meanwhile, a technique has been reported whereby cornea epithelial cells or conjunctival epithelial cells are cultured and organized on amnion from which the sponge layer and the epithelial layer have been removed such that cell fragments together with the amnion are used for implantation (JP Patent Publication (Kokai) No. 2001-161353 A). Since amnion has sufficient membrane strength and no antigenicity, it is convenient to use amnion as a support for cell fragments used for implantation. However, in accordance with the method disclosed in the above reference, it is impossible to arrange cells in fine patterns and to culture the cells.